Culture chamber and culture method

Abstract

Provided is a culture chamber that includes a plurality of recesses each formed of a bottom portion and an opening portion. The bottom portion has a hemispherical shape or a truncated cone shape. The opening portion is defined by a wall that surrounds an area from a boundary between the opening portion and the bottom portion to an end of each of the recesses, the wall having a taper angle in a range from 1 to 20 degrees. An equivalent diameter of the boundary is from 50 μm to 2 mm and a depth from a bottom of the bottom portion to the end of each of the recesses is from 0.6 or more times to 3 or less times the equivalent diameter, and the wall defining the opening portion forms a surface continuous to the bottom portion. An inclination of the continuous surface changes at the boundary.

What is claimed is: 
     
       1. A method for cell seeding, cell culture, replacement of a medium, and harvesting of cells, comprising:
 dispersing cells into a medium, a total number of the cells being greater than or equal to a number N of recesses of a culture chamber and less than or equal to a number obtained by multiplying the number N of the recesses by a value obtained by dividing a volume V 1  of a space defined by each of the recesses by a volume V 2  of cells to be seeded; and 
 adding the medium to the culture chamber; 
 wherein each of the recesses of the culture chamber is formed of a bottom portion and an opening portion, wherein: 
 
       the bottom portion comprises a hemispherical shape or a truncated cone shape,
 the opening portion is defined by a wall that surrounds an area from a boundary between the opening portion and the bottom portion to an end of each of the recesses, the wall comprising a taper angle of 1 to 20 degrees, 
 an equivalent diameter of the boundary is 50 μm to 2 mm and a depth from a bottom of the bottom portion to the end of each of the recesses is 0.6 or more times to 3 or less times the equivalent diameter, and 
 the wall defining the opening portion forms a surface continuous to the bottom portion, and an inclination of the surface continuous to the bottom portion changes at the boundary, and 
 wherein each of the recesses comprises a cell non-adhesive surface on which at least one polymer of a hydrophilic polymer chain is immobilized after a functional group is formed in the recess by a surface modification treatment method selected from the group consisting of a coating, corona discharge, UV ozonation, and a combination thereof, and the treatment is performed so that a water contact angle becomes 45 degrees or less. 
 
     
     
       2. The method according to  claim 1 , wherein one spheroid is formed in one space defined by each of the recesses. 
     
     
       3. The method according to  claim 2 , wherein the spheroid is allowed to grow. 
     
     
       4. The method according to  claim 2 , wherein the spheroid is differentiated and induced. 
     
     
       5. The method according to  claim 1 , wherein 60% or more of a total number of spheroids formed in the culture chamber have a diameter in a range of ±5% of an average spheroid diameter. 
     
     
       6. The method according to  claim 1 , further comprising agitating the medium, wherein the cells in the recesses are recovered by the agitating. 
     
     
       7. The method according to  claim 6 , wherein the agitating comprises shaking the culture chamber, sucking and discharging the medium, disposing a stirring blade in the culture chamber, or placing a stirrer in the culture chamber. 
     
     
       8. The method according to  claim 1 , further comprising replacing the medium at least once, wherein 20% or more of the medium is replaced during the replacing. 
     
     
       9. The method according to  claim 1 , further comprising immobilizing a poly(hydroxyethyl methacrylate) on the modified surface. 
     
     
       10. The method according to  claim 9 , wherein an area between two adjacent recesses is flat and a distance between the two recesses is 5 μm to 50 μm. 
     
     
       11. The method according to  claim 1 , wherein poly(hydroxyethyl methacrylate) is immobilized in each of the recesses. 
     
     
       12. The method according to  claim 11 , wherein an area between two adjacent recesses is flat and a distance between the two recesses is 5 μm to 50 μm. 
     
     
       13. A method of cell seeding, cell culture, replacement of a medium, and harvesting of cells comprising:
 dispersing cells into a medium, the number of cells being equal to or greater than a number n of recesses of a culture chamber and less than or equal to a number obtained by multiplying the number n of the recesses by a value obtained by dividing a volume V of each of the recesses by a volume v of cells to be seeded; 
 adding the medium to the culture chamber; 
 culturing the cells in the culture chamber for 12 hours or more to form a spheroid; 
 sucking 20% or more of the medium and then injecting the same amount of fresh medium into the culture chamber;
 allowing the spheroid to grow to a desired size; 
 
 agitating the medium to suspend the cells within the recesses in the medium; and 
 sucking the medium comprising the cells by a suction machine to recover the cells; 
 wherein each of the recesses of the culture chamber is formed of a bottom portion and an opening portion, wherein: 
 the bottom portion comprises a hemispherical shape or a truncated cone shape, the opening portion is defined by a wall that surrounds an area from a boundary between the opening portion and the bottom portion to an end of each of the recesses, the wall comprising a taper angle of 1 to 20 degrees, 
 an equivalent diameter of the boundary is 50 μm to 2 mm and a depth from a bottom of the bottom portion to the end of each of the recesses is 0.6 or more times to 3 or less times the equivalent diameter, and 
 the wall defining the opening portion forms a surface continuous to the bottom portion, and an inclination of the surface continuous to the bottom portion changes at the boundary. 
 
     
     
       14. The method according to  claim 13 , further comprising modifying a surface of each of the recesses by a surface modification treatment selected from the group consisting of a coating, corona discharge, UV ozonation, and a combination thereof to form a functional group within each of the recesses, wherein a water contact angle of the modified surface is 45 degrees or less. 
     
     
       15. The method according to  claim 14 , further comprising immobilizing a poly(hydroxyethyl methacrylate) on the modified surface. 
     
     
       16. The method according to  claim 15 , wherein an area between two adjacent recesses is flat and a distance between the two recesses is 5 μm to 50 μm. 
     
     
       17. The method according to  claim 13 , wherein each of the recesses comprises a cell non-adhesive surface on which a poly(hydroxyethyl methacrylate) is immobilized after a functional group is formed in the recess by a surface modification treatment method selected from the group consisting of a coating, corona discharge, UV ozonation, and a combination thereof, and the treatment is performed so that a water contact angle becomes 45 degrees or less. 
     
     
       18. The method according to  claim 17 , wherein an area between two adjacent recesses is flat and a distance between the two recesses is 5 μm to 50 μm.